With the availability of atomic scale structures of ribosomes, the critical task is to link ribosome structure with biological function. Our main focus has been to link ribosome structure with its critical functions during the translation elongation cycle. We consider these essential functions to be: distinguishing between cognate, near-cognate and non-cognate codons;maintenance of translational reading frame;and termination codon recognition. More recently we have expanded our studies to explore the interactions between ribosomes and Internal Ribosome Entry Signals (IRES elements). In targeting components of the ribosome, the focus is on core, evolutionarily conserved features of the Saccharomyces cerevisiae ribosome because it represents the most genetically and biochemically malleable model eukaryotic system available. This foundation has been built upon to develop a robust and synergistic combination of molecular genetic, biochemical, structural, and molecular modeling tools. This has enabled us to show that both the biophysical interactions between ribosomal proteins, rRNAs and tRNAs, and the biochemical properties of ribosome-associated enzymatic activities are critical for carrying out the biological functions enumerated above. On a broader scale, this research program is defining the allosteric communication pathways that connect and coordinate different functional centers of the ribosome with one another. Thus, the ribosome can be conceived as a model nanoscale machine and of ours as a reverse engineering program. To further define how ribosome structure influences function, we propose to further develop the program by: 1) refining our understanding of previously identified targets, 2) expanding the list of targets for analysis, and 3) refining and expanding our biochemical and molecular toolbox. The general strategy is to determine the effects of targeted mutations on yeast ribosome structure and function. This is broken down into three specific aims. Aim 1 will determine the effects of targeted mutations of Ribosomal Proteins (RPs) on yeast ribosome structure and function. This will use reverse genetics approaches involving targeting of specific, previously identified amino-acids to ribosomal proteins L2, L3, L10, and L11. The list of protein targets will also be expanded to include ribosomal proteins S15, and S18. Aim 2 seeks to determine the effects of targeted mutations of Ribosomal RNA (rRNA) on yeast ribosome structure and function. A reverse genetics approach will target specific rRNA bases based on i) their associations with known ribosomal functions, and ii) their interactions with ribosomal proteins as defined in preliminary studies and Aim 1. Aim 3 will determine the effects of targeted mutations of rRNA modification on ribosome structure and function characterizing the effects of rRNA modification mutants in yeast, mouse and human cells on translational fidelity. PROJECT NARRATIVE: Proliferating cells, be they embryonic cells busily creating new persons, T-cells fighting off infection, or cancer cells overwhelming the patient, absolutely require large numbers of highly accurate ribosomes to meet their needs for synthesis of new proteins. Ribosomes, the central component of this process, are complex biological nanomachines composed of many protein and RNA molecules, and the overall goal of the proposed research is to begin to understand how the atomic scale structure of the ribosome ultimately determines its function. A deeper understanding of the relationship between ribosome structure and function will aid the rational design of new classes of drugs designed to target a diverse array of clinical applications including antiviral and antibacterial agents, as well as drugs targeting a diverse array of cancers, developmental disorders, and other critical diseases afflicting society.